Many chemicals useful in industry are amphiphilic compounds which can be obtained by esterification or reverse hydrolytic reactions between hydrophilic compounds and second compounds, which may be hydrophobic. Illustrations of such reactions follow.
1) Esterification of a polyalcohol:
glycerol+free fatty acid.fwdarw.mono-glyceride+water PA0 glucose+free fatty acid.fwdarw.sugar-ester+water PA0 peptide+free fatty acid.fwdarw.peptide ester+water PA0 1-sn-monoglyceride=(S)-1-monoglyceride PA0 3-sn-monoglyceride=(R)-1- monoglyceride PA0 2-sn-monoglyceride=achiral
2) Esterification of a carbohydrate:
3) Esterification of an amino acid:
The amphiphilic products of such reactions are used as surfactants, emulsifiers, food additives, food components, and food substitutes as well as pharmaceuticals. Amino acid and peptide esters possess biological activities similar to those of lipoproteins found in nature, and are potentially useful as pharmaceuticals.
The reactions above are depicted in the direction of reverse hydrolysis, which will dominate only under low water conditions, i.e. in hydrophobic, non-polar solvent. Since the hydrophilic compound is not miscible with non-polar solvent, it is problematic to establish reaction conditions which are favorable to reverse hydrolysis. Moreover, most hydrophilic and hydrophobic compounds are immiscible in one another, and for many compound combinations there is no suitable solvent which would allow the mixing of both substrates. Consequently, esterifications of many hydrophilic compounds, such as glycerol, ethyleneglycol, polyols, and saccharides with long chain fatty acid residues, are difficult or impossible to achieve by traditional means in a batch or column reactor. For instance, the conventional industrial process for the esterification of glycerol with fatty acids is conducted at 200.degree.-250.degree. C. in the presence of an inorganic catalyst, often a toxic metal.
One class of amphiphilic products useful as emulsifiers includes long-chain monoacylglycerols or monoglycerides. Conventionally, they are produced by chemical alcoholysis of the corresponding triglycerides with two equivalents of glycerol (see FIG. 5B). As in industrial esterification, this type of reaction requires high temperatures (210.degree.-240.degree. C.) and the use of transesterification catalysts, usually toxic tin or lead compounds. The reaction product is an equilibrium mixture consisting of monoglyceride, glycerol as well as fatty acids and several contaminating by-products resulting from dehydration processes. In order to yield "pure" monoglycerides, as regioisomers, expensive purification steps are required leading to product yields of only about 40-50% based on the starting glycerides.
Alternatively, chemical multistep processes are reported in the literature based on isopropylidene-glycerol (Bear, E., Fischer, H.O.L., J. Am. Chem. Soc. 67, 2031 (1945)) or glycidol (German Patent DE-OS 2,338,462 (1973)).
In these methods, natural starting materials cannot be employed. All the above methods require the synthesis of suitable starting materials and/or protection/deprotection steps. Thus, the above described conventional synthetic methods do not provide convenient means to obtain chemically or isomerically pure monoglycerides.
A potential alternative is offered by biocatalysis utilizing enzymes (for an overview, see Wong, C. H., Science 244, 1145-1152 (1989)). In the chemical reactions discussed herein, both of the compounds changed in the reaction are considered substrates for the enzyme (Wong, supra). In order for enzymatic reverse hydrolysis to be made practical for industrial use, the above mentioned problems of substrate/solvent immisibility must be overcome.
Catalysis of esterification reactions by the enzyme lipase has been described. It was reported that, in a reaction between glycerol and free fatty acids, mycelial lipases catalyzed the formation of ester bonds at positions 1 and 3 of glycerol to form mono- and diglycerides (Tahoun, M. K. et al., Enzyme Microb. Technol. 8, 429-432 (1986)). Reactions were carried out in aqueous medium, with dispersion promoted by stirring.
Lipases have also been used in the esterification of saccharides with fatty acids (Bjorkling, F. et al., J.C.S. Chem. Commun., 934-935 (1989); Adelhorst, K. et al., Synthesis, 112-115 (1990)). The reactions were conducted without added solvent since the two substrates were somewhat soluble in one another. Water produced in the reactions was removed in vacuo.
For the preparation of monoglycerides, enzymatic multistep processes have been reported based on the starting compounds isopropylideneglycerol (Omar, I. C., Sacki, H., Nishio, N., Nagai, N., Biotechnol. Lett. 11, 161 (1989)) or glycidol (Muller, C., Austin, H., Posorske, L., Gruzalez, I., Novo Industri Publication A-05991). These enzymatic methods have the same limitations as the chemical multi-step processes described above.
In order to increase the rate of reaction and yield, lipase-catalyzed esterification in aqueous medium has been carried out in membrane bioreactors. In the membrane bioreactor system, reverse hydrolysis is promoted through the use of microporous hydrophobic membranes which physically separate the glycerol/water/lipase solution from the fatty acid substrates (Yamane, T. et al., Ann. N.Y. Acad. Sci. 434, 558-568 (1984); Hoq, M. M. et al., Agric. Biol. Chem. 49, 335-342 (1985)). The fatty acids penetrate the micropores of the hydrophobic membrane sufficiently to participate in the reaction at the interface of the membrane and the aqueous solution; the resulting amphiphilic products remain in the aqueous compartment.
Another approach requires covalent linkage of substrate to a solid support in order to increase the exposure of substrate surface area to reactive groups. Enzymatic peptide synthesis has been promoted in aqueous medium by ester or amide bond linkage of amino acid substrate to activated silicagel (Koennecke, A. et al., Monatsh. Chem. 113, 331-337 (1982); Koennecke, A. et al., Monatsh. Chem. 111-117 (1985)).
Lipase catalyzed reactions have also been carried out in reverse micellar systems or microemulsions. These systems were designed to promote the exchange of fatty acid side chains on triglycerides. The dispersion of triglycerides in polar solvent was achieved through emulsification with surfactants. When the hydrophilic substrate glycerol was substituted for aqueous buffer in the emulsion system, no ester product was formed. (Holmberg, K. et al., JOACS 65, 1544-1548 (1988); Zaks, A., JAOCS 66, 484 (1989)).
Peptide synthesis was reportedly catalyzed by chymotrypsin in an organic solvent, hexane, in which neither the substrates nor the enyzme were soluble (Kuhl, P. et al., Tetrahedron Lett., 31 5213-5216 (1990)). Product yields were inversely proportional to hexane concentration, and fell to zero at moderate concentrations of hexane. Although the authors could offer no conventional explanation for their results, they speculated that the undissolved substrates promoted the reaction through direct particle-particle contact.
Glycoside transfer is another type of reaction which may be enzymatically catalyzed. Products of glycoside transfer include complex oligosaccharides, glycoproteins, and glycolipids. Glycosyltransferases are not yet readily available, but the corresponding hydrolases, i.e. glycosidases (Table 1, category 3.2.1) are commercially available. The glycosidase, .beta.-galactosidase, has been employed to catalyze reactions between saccharides and alcohols. The reported yields were low (25-35%) because the reactions were carried out in an aqueous medium in which the initially formed products were partly decomposed by the hydrolytic action of the enzyme. The reported procedures could not be carried out in non-aqueous medium because of immiscibility of substrates with non-polar solvents.
The problem of enzyme insolubility in organic solvents has been addressed by the adsorption of peptidases and lipases to solid supports such as celite and silicagel (Ferjancic, A. et al., Appl. Microbiol. Biotechnol. 36, 651-657 (1990); Schuch, R. et al., Appl. Microbiol. Biotechnol. 30, 332-336 (1989); Eigtved, P. et al., Proc. World Conf. Biotechnol. Fats and Oils, AOCS, 134-137 (1987); Yamanaka, S. et al., Methods in Enzymol. 136, 405-411 (1987)). Amounts of silicagel or celite sufficient to adsorb catalytic amounts of enzyme were either added to the reaction mixture, or, more commonly, were purchased with the enzyme pre-adsorbed onto the support. With stirring, the adsorbed enzyme was dispersed in the hydrophobic solvent. The hydrophilic substrate, however, remained ineffectively dispersed in the solvent which limited the rate of reaction and final product yield.
The hitherto reported phosphorylation of biologically important molecules like glycerol, dihydroxyacetone, enolpyruvate, monosaccharides, and nucleosides was dependent on biological phosphorylating agents, usually adenosine triphosphate (ATP). These reactions reportedly are catalyzed by such enzymes as glycerol kinase and hexokinase, and are carried out in aqueous medium. Such processes are costly because ATP is expensive, is needed in molar amounts, and therefore must be recycled. Recycling of ATP involves a number of additional enzymatic steps and auxiliary reagents.
In addition to limitations in yield and rate of reaction, the published methods leave unsolved the problem of producing isomerically and enantio-merically pure products such as 1-monoglycerides. When glycerides are produced in the above described enzymatic methods, mixtures of mono-glycerides with di- and tri-glycerides are obtained. When monoglycerides are isolated from the product mixture, they are always mixtures of regioisomers and stereoisomers. Monoglycerides are classfied according to their absolute configuration as follows.
The "sn-" nomenclature is a classification which is exclusively used for glycerides. In the conventional production of monoglycerides as described above, generally mixtures of racemic 1-monoglycerides and achiral 2-glycerides are produced. Such mixtures could be termed as consisting of: (R.S)-1-monoglyceride+2-monoglyceride. Purification steps are required to yield chemically pure monoglycerides as mixtures of regioisomers from which each regioisomer must be separated by tedious chromatographic processes. These processes necessarily operate on a very small scale and are not practical for industrial production.